Therapeutic efficacy of spirulina against ovalbumin and cigarette smoke‐induced asthma‐specific stress biomarkers in Sprague–Dawley rats

Abstract Due to the high prevalence of allergies and asthma, awareness about allergens and therapeutic use of functional foods and nutraceuticals have gained immense attention. Spirulina powder is being used as health‐boosting and antioxidant agent against several ailments owing to its unique nutritional profile. Considering its antioxidant role, the current study was focused on exploring therapeutic role of spirulina against stress biomarkers in asthmatic model. To assess the therapeutic efficacy of spirulina against allergic asthma‐specific oxidative stress biomarkers, a model feed trial was conducted and rats were divided into four groups (n = 10). G0–I (negative control), G0–II (positive control), whereas GI (spirulina) and G2 (salbutamol) served as treatment groups. Salbutamol is a chemical compound which is used in several antiallergic medicines because it works as bronchodilator. G2 group was given salbutamol for comparison of results. For asthma induction, rats were given intraperitoneal injection of ovalbumin on 7th, 14th, and 21st day. Treatment groups were given spirulina powder (500 mg/kg body weight) and salbutamol (1 mg/kg), respectively, after the induction of asthma. All three asthmatic groups were also exposed to cigarette smoke daily along with respective treatment for 4 weeks. Asthma induction caused an increase in total cell count in bronchioalveolar fluid (BALF), while spirulina treatment reduced total cells in BALF by 33.50% and salbutamol by 41.7%. Level of interleukins (IL) like IL‐4 decreased by 33.32% & 48.56% in G1 and G2. Similarly, IL‐5 and IL‐13 levels reduced by 40.9% & 49.9% and 18.62% & 38.02%, respectively, in G1 and G2. Serum levels of Immunoglobin‐E (Ig‐E) declined by 29.70% and 52.82%, while histamine levels were 26.23% & 45.58% less at the end of study in comparison to positive control. Moreover, histological analysis of lung tissue revealed that both spirulina and salbutamol effectively reduced ovalbumin and cigarette smoke‐induced moderate to severe necrosis, architectural changes, and congestion. It was concluded that salbutamol showed better results however, spirulina also effectively reduced mild to moderate allergic symptoms in dose‐dependent manner. Nutraceutical and functional foods are considered helpful in mitigating oxidative stress‐mediated health problems. Spirulina has its unique nutritional profile including phycobiliproteins, phytochemicals, and antioxidant vitamins which make it useful against several ailments. Considering its antioxidant role, current study was focused on exploring therapeutic efficacy of spirulina against stress biomarkers in asthmatic model. Outcomes of present research also demonstrated beneficial effect of spirulina in modulating allergic symptoms. In this regard, ancient concept of “medicine food homology” can be implemented and spirulina can be incorporated in food for additional benefits. However, further research regarding safety aspects is needed for its use in clinical practice for humans.


| INTRODUC TI ON
According to the traditional definition, an allergy is a set of adverse effects that are triggered by the secondary immune response while interacting a foreign antigen which is generally harmless (Dhudasia et al., 2021). Allergies are complex disorders associated with various factors including genetic and environmental, etc. Their association can be determined by evaluating the expression of disease and its various phenotypes (Pawankar et al., 2011). Allergic disorders are prevalent among all age groups and are common worldwide resulting a major health burden. In this context, various mechanisms are involved but immunoglobin-E (Ig-E)-mediated reactions are most prominent. Asthma, food allergy disorders, rhinitis, gastrointestinal disorders, and atopic dermatitis are categorized as major chronic allergic disorders that may cause irreversible impairments to effected organ (Dhudasia et al., 2021). Asthmatic response of lung to the inhaled allergen is often described by two different phases, that is, early-phase response (EPR) and late-phase response (LPR). In EPR, allergic symptoms occur within 1 h of exposure to allergens whereas in LPR, symptoms occur several hours later with prolonged persistence and severity (Drazdauskaitė et al., 2021;Gandhi et al., 2017).
Pathophysiological mechanism of asthma involves oxidative stress and increased exposure to reactive oxygen species (ROS) (Czerska et al., 2016). ROS are produced extrinsically by environmental factors, that is, smoking and air pollutants and internally by metabolic reactions . These overproduced ROS are balanced by endogenous antioxidant defense system involving enzymatic antioxidants like SOD, CAT Gpx, etc., while non-enzymatic antioxidants include vitamin C, E, and zinc, etc. (Mittal et al., 2014;Ramzan et al., 2021). Studies have reported significant association of these oxidative stress biomarkers with the characterization of asthma, especially in adults (Czerska et al., 2016;Ramzan et al., 2021).
Interaction with allergens (animal fur, pollens, molds, dust mites, and cockroaches) and non-allergens (exercise, smoke, cold air, and infections) causes differentiation of T helper cell in T cell type-2 which further cause cascade of allergic responses. Hence, in response to an increase in Th2 levels, cells release inflammatory cytokines like interleukins . These cytokines promote inflammation by initiating eosinophilic response resulting in an increased Ig-E production which binds to receptors on mast cells and initiates the release of inflammatory mediators like histamine and leukotrienes (Bulowa et al., 2018;Fleischer et al., 2021).
In the United States, the use of natural products, dietary supplements, and vitamins as an additional treatment shows 40% of the conventional therapies (Costa et al., 2010;Ventola, 2010). According to various studies, alternative medications associated with natural products by involving multiple immunomodulatory biochemical pathways could reduce the pathogenesis of most diseases (Jiang et al., 2021;Panossian et al., 2021;Shaw et al., 2021;Xing et al., 2021). A stepby-step approach is required to manage allergic diseases. Shared care plan and self-management lead to improvement in asthma outcomes (Wilson et al., 2010). Furthermore, knowledge regarding proper inhaler strategies, avoidance from irritants & allergens, and medication compliance are important for each patient suffering from asthma.
For the last 5000 years, products derived from plants have been useful for the treatment of asthma known as traditional or folk medicines against asthma. Plants have many secondary metabolites that possess bronchodilator and anti-inflammatory properties, thereby enhancing anti-asthmatic activities. Terpenoids, flavonoids, and phenolic acids are the main constituents that increase anti-asthmatic properties. Nowadays, people are more interested in healthy foods for disease prevention which contain high nutritional value and play a vital role in promoting human health (Dawoud, 2016). Beneficial microalgae have been a part of human diet and animal feed since decades. It grows in fresh and marine water as filamentous organism.
Notably, a significant amount of nutrients is present in these microorganisms including essential amino acids and fatty acids along with many other bioactive components (Abdulmumin, 2013). Spirulina is a spiral-shaped threadlike alga that belongs to family Oscillatoriaceae. It is naturally found in pollution-free, mineral-rich, alkaline, and high pH water. It flourishes in alkaline water lakes where it is not possible for other micro-organisms to survive (Michael et al., 2019). Spirulina acquires its blue-green color due to the variety of pigments having their functional properties like chlorophyll a, β-carotene, zeaxanthin, and considered helpful in mitigating oxidative stress-mediated health problems. Spirulina has its unique nutritional profile including phycobiliproteins, phytochemicals, and antioxidant vitamins which make it useful against several ailments. Considering its antioxidant role, current study was focused on exploring therapeutic efficacy of spirulina against stress biomarkers in asthmatic model. Outcomes of present research also demonstrated beneficial effect of spirulina in modulating allergic symptoms. In this regard, ancient concept of "medicine food homology" can be implemented and spirulina can be incorporated in food for additional benefits. However, further research regarding safety aspects is needed for its use in clinical practice for humans.

K E Y W O R D S
asthma, bronchoalveolar fluid, cigarette smoke, Ovalbumin, spirulina, stress biomarkers phycobiliproteins (C-phycocyanin, A-phycocyanin, and phycoerythrin) which acts as a free radical scavenger and reduces inflammation by correcting function of antioxidant enzymes present in the body (Arashiro et al., 2020). It has gained much attention as nutraceutical for its immunity-boosting effects (Mathur, 2019). Even though spirulina comprises good nutritional composition, its use and cultivation in Pakistan are very limited. Several researchers have demonstrated its antioxidant and anti-inflammatory potential (Asghari et al., 2016;Farag et al., 2016;Lechuga Morente, 2021;Masuda & Chitundu, 2019;Simon et al., 2018;Soltani et al., 2012) however, studies regarding its antiallergic effects are limited. Therefore, the present study was designed with the aim to check antiallergy and immunomodulatory effects of spirulina with special references to asthma.

| Hypothesis
Spirulina powder may play an effective role in attenuating asthmaspecific oxidative stress biomarkers. water at 23-25°C. After 1 week, the rats were randomly divided into four treatment groups (n = 10) to check antiallergic potential of spirulina powder. Purposely, the G 0-I was fed on normal diet and served as negative control whilst G 0-II was dependent on normal diet along with induction of asthma to serve as positive control. Whereas G I and G 2 were given spirulina powder (500 mg/kg body weight) and salbutamol (1 mg/kg), respectively after the induction of asthma. Later three groups were also given cigarette smoke daily during the treatment period in Chamber (25 cm high, 40 cm wide, and 30 cm long). Two cigarettes were placed in a small open hole at one side of glass chamber.

| Bioevaluation Trials
This set-up produced side-stream smoke that provoked the passive smoke or environmental tobacco smoke exposure. Trial was carried out for 7 weeks, first 3 weeks for asthma induction and next 4 weeks for treatment. After the set time, overnight fasted rats were decapitated, blood, serum, and tissue samples were collected to check the efficacy of spirulina powder against oxidative stress biomarkers.

| Induction of bronchial asthma
Asthma was induced in rats by giving them intraperitoneal injection of 1 mg ovalbumin and 20 mg aluminum hydroxide Al (OH) 3 mixed in phosphate buffer saline (pH:7.4) on 7, 14, and 21st day.
All three groups (except negative control group) were exposed to cigarette smoke (to enhance allergic symptoms related to asthma) on daily basis along with respective treatment for 4 weeks. (Abdеlaziz et al., 2018;Ezz-Eldin et al., 2020). (Table 1)

| Blood sampling and analysis
After the completion of trial, rats were anesthetized and sacrificed.
Blood and serum samples were collected in different tubes considering analyses to be performed.

| Hematology
The effect of spirulina powder on oxidative damage of cells caused by allergen and cigarette smoke was assessed by performing white blood cells count (Qinghua et al., 2016). Purposely, a hematology analyzer and Soluplastin-kit (Wiener-Lab, Argentina) were used.
For serum collection, coagulated blood samples were centrifuged at 4000 rpm for 15 min. The upper serum layer was cautiously collected and kept at −40°C for analyses (Ezz-Eldin et al., 2020).

| Bronchoalveolar lavage fluid (BALF) collection
Bronchoalveolar lavage is a useful method for the identification of pathologies associated with lungs. Bronchoalveolar lavage(BAL) is a clinical practice in which Fluid is squirted into the lung and then recollected for WBC estimation. High amount of white blood cells present in lungs indicates the severity of lungs inflammation. Purposely, trachea and lungs were exposed by dissection of the chest cavity.
For BALF collection, PE-90 polyethylene tube linked to a needle hub was gradually introduced into the trachea and 5 ml of cold phosphate-buffered saline (PBS) was injected. The PBS was allowed to stay in the lungs for 30 s, while the thoracic area was slightly massaged and suctioned gradually through the needle hub. This procedure was done three times in all groups and BALF was collected followed by centrifugation of fluid for 10 min. Afterward, BAL cells were suspended in 1 ml of saline, stained with Turk's solution, and calculated by a hemocytometer (Song et al., 2010).

| Histopathological examination
Histological examination of lungs and liver tissues of rats was performed by following the protocols of Kafle et al. (2018). At the end of the bio-efficacy trial, lungs and liver of rats were removed followed by fixation in 10% formalin in saline. After 72 h of fixation, the sections were cut and embedded in paraffin wax for dehydration. After cooling, the paraffin blocks were decalcified in water for 40-50 min followed by washing and kept frozen till trimmed. Five-micrometerthick pieces were taken and stained with hematoxylin and eosin on glass slide to assess degree of inflammation. The stained slides were observed under light microscope for digital image collection.

| Statistical analysis
The obtained data from each parameter were subjected to statistical analysis to check the level of significance. Completely Randomized Design was applied by using Statistix 8.1 software. Furthermore, Tukey's HSD test was applied to analyze the differences in means (Montgomery, 2008).

| Physical signs of allergy and asthma progression
After first injection of ovalbumin, no prominent changes were observed in the behavior of rats. No significant activity loss, hair loss, or irritation in nose were observed. On the 21st day of study (last ovalbumin injection), a gradual loss of activity was recorded. Rats of negative control group were active, while reduced activity was noticed in the later three groups. Moreover, the forced breathing rate was higher in asthmatic rats (G 0-II , G 1 , and G 2 ) than negative control (G 0-I ) alongside an increase in fluid intake. Non-substantial hair loss was also observed in some rats. After 1 week of last ovalbumin injection, rats were given respective treatments along with cigarette smoke daily. During smoke exposure, mostly rats were calm at initiation and subsequently start sleeping as exposure time increased. This behavior was noticed on daily basis. Within 1 week of treatment and cigarette smoke exposure, significant hair loss and decline in activity were observed in the asthmatic rats.
An increase in fluid intake was also evident. All these indicators were increasing with every passing week in positive control (G 0-II ).
Symptoms were also present in G I (spirulina treatment) and G II (drug treatment) however, level of severity was less as compared to G 0-II ( Table 2).

| Leukocyte's indices
Results of the present study inferred an increase in total WBCs and eosinophils and a reduction in serum lymphocyte count of asthmatic rats as compared to negative control. All these variations are in favor of asthma induction in animal model. Besides, all asthmatic rat groups exhibited significant (p < .05) differences in leukocytes in-

| Asthma-specific oxidative stress biomarkers
Means regarding asthma-specific oxidative stress biomarkers in serum (histamine, Ig-E, IL4, 5, and 13) of all treatment groups are expressed in Table 4. Furthermore, all asthmatic rat groups exhibited significant (p < .05) differences in asthma-specific oxidative stress biomarkers. Levels of stress biomarkers conspicuously varied among normal and asthmatic rats as these are released with the onset of disease or allergic conditions. Therefore, com-

| Total cell counts in BALF
Mean values pertaining to total cell count in the bronchoalveolar fluid (BALF) are displayed in Figure 1. The analysis depicted momentous differences among the groups of asthmatic rats. Findings   infections (Shakeri & Boskabady, 2017). Thus, the rise in the eosinophils level is a major feature of allergic asthma leading to many inflammatory responses (Nair & Prabhavalkar, 2021). of normal rats were recorded as 3.48 ± 0.79 Cells × 10 5 /ml which increased to 5.33 ± 2.46 Cells × 10 5 /ml attributed to allergy progression. Infiltration of cells in lungs was 53.16% higher compared to normal rats. In reference to effect of drugs, the trend of present outcomes is in accordance with recent findings of two researchers Nair and Prabhavalkar (2021). They reported that salbutamol and dexamethasone (0.5 and 0.1 mg/kg) effectively reduced total and differential cell count in BALF of treatment groups. Total cell counts decremented 70% with combined drug treatment. Furthermore, saffron protected oxidative stress along with salbutamol by reducing 45% cell infiltration in lungs. Similarly, another group of peers reported the same effects of asthma induction. Ovalbuminsensitized rats showed a marked incline in total and differential cell count in BALF. Eosinophils, macrophages, and neutrophils numbers reduced substantially in treatment groups receiving (42.5 mg/kg) fruit extract. Total cell count increased from 7.00 ± 0.52 × 10 5 to 11.17 ± 0.87 × 10 5 cells/ml attributed to asthma induction, whereas antioxidants components of fruit extract lessened total cell count to 8.11 ± 0.45 and dexamethasone (drug) to 4.83 ± 0.48 × 10 5 cells/ mL (Arora et al., 2016). Likewise, outcomes of Thakur et al. (2019) revealed total cell count in BALF of normal and asthmatic rats 9 × 10 3 cells/mm 3 and 15 × 10 3 cells/mm 3 , respectively. Cell count in BALF elevated up to 66.6% by asthma induction. Previously, Gholamnezhad et al. (2014) assessed effect of Fluticasone (corticosteroid) and Salmeterol (β2-agonist) on total WBC count in BALF.

TA B L E 4 Asthma-specific biomarkers of oxidative stress in experimental rats
Rats were administered both drugs during and after sensitization period. Normal cell level in BALF was 500, while Control and treatment groups showed 3000, 1000, and 1500 cell count/ml increment in BALF. Results demonstrated 66.60% and 50% reduction in total cell count due to treatment. Allergy induction increased overall ce0ll count in BALF up to 50-70% however, treatment with drugs showed a decline of 45%-70%. Research findings also indicated that treatment of allergic disorders with antioxidant-rich plant foods have also displayed prophylactic properties as proclaimed by current exploration.
Generally, autophagy is conducive to cells' existence, keeping the homeostasis by eliminating necrotic organs and proteins in response to environmental stress inducers like hypoxia, temperature fixation, and infectious pathogens. Oxidative stress causes excessive autophagy that can cause adverse effects like cell death. It is an underlying factor in pathogenesis of various diseases, the pathological mechanism of allergic asthma starts with the differentiation of T cells to allergen-specific T helper 2 cells, followed by the production of immunoglobulin E (Ig-E) . It is an allergen-specific immunoglobulin that attaches to its receptors ( Earlier findings have also shown that cigarette smoke could exacerbate ovalbumin-induced asthma and markedly enhance the expression of IL-4 and IL-13 genes (Sun et al., 2021). Glucocorticoids and β2-agonists are mainly used in modern medicine to treat allergic asthma. However, long-term use of glucocorticoids is prone to resistance . Besides, several marine algae have demonstrated role in blocking binding of Ig-E to its receptors on mast cells and release of inflammatory factors like interleukins and histamine (Sugiura et al., 2008  and edema was also reduced. Likewise, another investigation declared that treatment with R-phycocyanin from algae for 48 h suppressed ovalbumin-induced airway hyperresponsiveness. There were less infiltrated cells in bronchial region and R-phycocyanin was able to reduce airway inflammation caused by allergens (Chang et al., 2011). In reference to antiallergic effects of drug, Nair and Prabhavalkar (2021) reported that no significant changes were observed in normal control. However, positive control group demonstrated mild eosinophil and moderate to severe leukocyte infiltration. In case of treatment groups, normal parenchyma of lungs was preserved and less infiltration of cells in bronchial region was reported.

| CON CLUS ION
Conclusively, ovalbumin sensitization initiated allergy-like symptoms and cigarette smoke exposure played pivotal role in causing oxidative stress and enhancing allergic reactions. Both spirulina and salbutamol alleviated levels of oxidative stress biomarkers. However, salbutamol showed better results in mitigating asthma-specific oxidative stress biomarkers, while spirulina is also effective in reducing overall oxidative stress caused by allergy and cigarette smoke in dose-dependent manner primarily due to its bioactive components.
The histological analysis of lung and liver tissues expounded that both spirulina and salbutamol effectively reduced ovalbumin and cigarette smoke-induced moderate to severe necrosis, architectural changes, and congestion. Therefore, it is concluded that spirulina may have protective role against oxidative stress-induced cellular damage.

ACK N OWLED G EM ENTS
The authors are thankful to the Higher Education Commission, Pakistan, for providing funds under the project "Indigenous 5000 Fellowship Program." The authors are also thankful to the Faculty of Food, Nutrition and Home Sciences, University of Agriculture, for providing the technical support.

FU N D I N G I N FO R M ATI O N
The work has been sponsored by the Higher Education Commission under the project "Indigenous 5000 Fellowship Program."

CO N FLI C T O F I NTE R E S T
There is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data used to support the findings are included within the article.